JPWO2006126682A1 - Vaccine for prevention and treatment of Alzheimer's disease - Google Patents

Abstract

Provided are a vaccine for the prevention and treatment of Alzheimer's disease that is safe and easy to use with few side effects and a method for producing the same. Unprecedented safety due to the action of the antibody induced by administration of the product by expressing the Aβ molecular species, which are constituents of the senile plaque causing Alzheimer's disease, on the surface layer of bacteria exemplified by lactic acid bacteria In addition, an inexpensive oral type vaccine for the prevention and treatment of Alzheimer's disease is made possible.

Description

  The present invention relates to ethical drugs. Specifically, the present invention relates to a vaccine for the prevention and treatment of Alzheimer's disease, which accounts for about half of senile dementia that increases with aging, a method for producing the vaccine, and a vaccine antigen expression vector used in the production method.

  About 10% of elderly people over the age of 65 in Japan are reported to have senile dementia. One of the two major causes of senile dementia is Alzheimer's disease, accounting for about 50%. Furthermore, it is said that one in every two people over the age of 85 suffers from Alzheimer's disease or mild cognitive impairment, which is the preceding stage, which has become a major social problem not only in Japan but also in the West. The number of patients is reported to be about 15 million worldwide and more than 1.5 million in Japan.

  Currently, there are two commercially available Alzheimer's disease drugs, Donepezil, developed by Eisai Co., Ltd. and memantine, developed by Merz Co., both of which have the effect of delaying the progression of the disease. It cannot be cured.

  As a pathological hypothesis of Alzheimer's disease, an amyloid cascade hypothesis that is caused by the formation of senile plaques due to aggregation / deposition of amyloid β (hereinafter sometimes abbreviated as Aβ) is promising. Based on this hypothesis, vaccine (immunotherapy) is attracting attention as a new treatment for Alzheimer's disease. The purpose of this vaccine therapy is to administer Aβ as a vaccine and produce antibodies against it in the body to remove senile plaques and prevent aggregation of deposited Aβ and prevent neuronal loss It is something to try.

  Elan's Schenk et al. First reported that the formation of senile plaques decreased when Aβ was subcutaneously administered together with an adjuvant to an animal model mouse of Alzheimer's disease (see Non-Patent Document 1). . Based on this data, a clinical trial (AN-1792) was started for Alzheimer's disease patients by Elan Wyeth. However, side effects of meningoencephalitis were reported and discontinued in 6% (18/298) of patients in phase II of the clinical trial.

  On the other hand, Tahira of the National Institute for Longevity Medicine centered on the fact that antibody production is induced by using the intestinal tract immune system by oral vaccine, but cellular immunity is hardly induced. Adeno-associated virus (AAV) vector As a result of examining the effect by incorporating secreted Aβ cDNA into Alzheimer mice and orally administering it to the Alzheimer mouse, an antibody against Aβ was induced, and the antibody against Aβ in serum persisted for 6 months with a peak at 4 weeks (Patent Literature). 1). 13-month-old amyloid precursor protein (hereinafter abbreviated as APP) transgenic mouse brain tissue was examined in detail by immunostaining. As a result, amyloid deposition and senile plaque formation were clearly compared with control in the treated mouse brain. It was decreasing. This study shows that Alzheimer's disease can be treated in a way that is less burdensome for patients with oral administration. However, even though it is administered orally, adeno-associated virus is highly infectious to humans and has pathogenicity. Moreover, it takes a lot of money and time to culture adeno-associated virus and purify it as a vaccine. In other words, a vaccine using a viral vector is not ideal in terms of safety and cost.

JP 2005-021149 A Schenk D. et al., 1999 Jul 8, Nature; 400 (6740): p.173-7, "Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse"

  An object of the present invention is to provide a vaccine for the prevention / treatment of Alzheimer's disease that can be provided for the prevention / treatment of Alzheimer's disease, which has become a social problem, and that can be used in a safe and simple administration form with few side effects. .

  Another object of the present invention is to provide an expression vector for expressing an Aβ molecular species as a vaccine antigen on the surface of a microorganism, which is used in the preparation of a vaccine for the prevention / treatment of Alzheimer's disease, and the expression Providing a host microorganism transformed with a vector. The present invention also provides a method for inducing an anti-Aβ antibody by administering a microorganism expressing an Aβ molecular species to an individual, and a method for preventing and / or treating Alzheimer's disease based on the method for inducing the anti-Aβ antibody. In the present specification, the Aβ molecular species is a general term for a group of partial polypeptides including Aβ full-length molecules and Aβ humoral immunity induction sites based on the molecules.

  As a result of diligent research to solve the above problems, the inventors of the present application aimed to induce antibodies against Aβ, which is considered to be the main etiology of Alzheimer's disease, and obtained DNA encoding a fragment containing a humoral immunity-inducing site of Aβ. Based on this, an expression vector capable of expressing the peptide on the surface of the microorganism is constructed, and when a suitable microorganism is transformed using the expression vector, the desired peptide can be expressed on the surface layer. It came to achieve this invention which can provide the vaccine for prevention and treatment of Alzheimer's disease. In particular, when a lactic acid bacterium is selected as the host microorganism, a preferable vaccine that can be administered orally can be provided.

  If the Alzheimer's disease vaccine using lactic acid bacteria which is the optimal embodiment according to the present invention is used, it becomes possible to take preventive and therapeutic drugs for Alzheimer with the same feeling and convenience as conventional foods of lactic acid bacteria. Considering that this disease is particularly common among the elderly, oral administration that can be ingested easily and the realization of low cost will bring great benefits to society from the perspective of improving patient quality of life and the medical economy in the aging society. It can be said that the invention is given.

FIG. 1 is a gene map of the recombinant expression vector pHCEIILB-pgsA-L-Aβ42 for displaying the surface layer of lactic acid bacteria cells of Aβ42 of the present invention.

FIG. 2 shows the results of Western blotting of lactic acid bacteria transformed with the recombinant expression vector for expressing Aβ42 of the present invention, pHCEIILB-pgsA-L-Aβ42, showing surface expression of Aβ42 in the cell membrane fraction. M: molecular weight marker, C: lactic acid bacteria (control) whole cell fraction not having Aβ gene, 1: transformant whole cell fraction, 2: transformant cytoplasm fraction, 3: transformant cell membrane fraction.

FIG. 3 is a diagram showing the expression on the surface layer in the cell membrane fraction of Aβ42 of lactic acid bacteria transformed with the recombinant expression vector pHCEIILB-pgsA-L-Aβ42 for presenting Aβ42 of the present invention, based on Facs scan. Yes.

FIG. 4 is a gene map of the recombinant expression vector pHCEIILB-pgsA-L-AβN-20 for display of AβN-20 of the present invention.

FIG. 5 shows the results of Western blotting of lactic acid bacteria transformed with the recombinant expression vector pHCEIILB-pgsA-L-AβN-20 for display of AβN-20 of the present invention. Surface expression in the cell membrane fraction of AβN-20 Indicates. M: molecular weight marker, C: lactic acid bacteria (control) whole cell fraction not having Aβ gene, 1: transformant whole cell fraction, 2: transformant cytoplasm fraction, 3: transformant cell membrane fraction.

FIG. 6 is a diagram showing surface expression in the cell membrane fraction of AβN-20 of lactic acid bacteria transformed with the recombinant expression vector pHCEIILB-pgsA-L-AβN-20 for presenting AβN-20 of the present invention. Based on the Facs scan.

FIG. 7 shows the measurement of antibody titer in mouse serum when lactic acid bacteria transformed with pHCEIILB-pgsA-L-AβN-20 (AβN-20 expression vector) obtained in Example 2 were orally administered to mouse C57BL6 strain. Shows the results of the ELISA method. Panel (A) and panel (B) show the results when 16 amino acid residues and 42 amino acid residues were immobilized on the N-terminal side of Aβ, respectively.

FIG. 8 shows the measurement of antibody titer in mouse serum by the ELISA method when lactic acid bacteria transformed with pHCEIILB-pgsA-L-Aβ42 (Aβ42 expression vector) obtained in Example 1 were orally administered to mouse C57BL6 strain. The results are shown. Panel (A) and panel (B) show the results when 16 amino acid residues and 42 amino acid residues were immobilized on the N-terminal side of Aβ, respectively.

  An expression vector for producing an Aβ molecular species on the surface of a microorganism exemplified by lactic acid bacteria as a particularly preferred embodiment according to the present invention is a form capable of functioning a DNA encoding a fragment containing a humoral immunity induction site of Aβ. Thus, the peptide can be expressed on the surface of the microorganism. Here, “comprising in a functional form” means in a manner that allows expression of the transgene (DNA) under the control of appropriate regulatory elements (eg, promoters, enhancers, transcription terminators, etc.) It means that the transgene is inserted into the vector.

  It is considered that there are a plurality of humoral immunity induction epitopes of Aβ. To prepare a fragment containing the epitope, for example, if the epitope is present in the region of 4th to 10th amino acids of Aβ (Aβ4-10) The expression vector for producing them on the surface of the microorganism will contain a gene encoding Aβ4-10.

  The nucleotide sequence of DNA encoding this amino acid sequence is not particularly limited, and examples thereof include the 12th to 30th nucleotides in the nucleotide sequence represented by SEQ ID NO: 4. In addition, by using codons having the same amino acid sequence but different base sequences, the expression level can be increased by using a codon preferred by a microorganism such as lactic acid bacteria to be used.

  According to a preferred embodiment of the present invention, the antigen peptide fragment comprises the first to 42 amino acids (Aβ1-42) of the Aβ peptide. The amino acid sequence of Aβ1-42 is exemplified by the amino acid sequence represented by SEQ ID NO: 5, and therefore the antigen peptide fragment includes the amino acid sequence represented by SEQ ID NO: 5. The nucleotide sequence of DNA encoding this amino acid sequence is not particularly limited, and examples thereof include the nucleotide sequence represented by SEQ ID NO: 4. Further, by using codons having the same amino acid sequence but different base sequences, codons preferred by microorganisms such as lactic acid bacteria to be used can be used.

  According to another preferred embodiment of the present invention, the antigen peptide fragment comprises amino acids 1 to 20 (Aβ1-20) of Aβ peptide. Examples of the amino acid sequence of Aβ1-20 include those containing amino acids 1 to 20 in the amino acid sequence represented by SEQ ID NO: 5. The nucleotide sequence of the DNA encoding this amino acid sequence is not particularly limited, and examples thereof include the nucleotide sequence represented by SEQ ID NO: 4. Further, by using codons having the same amino acid sequence but different base sequences, codons preferred by microorganisms such as lactic acid bacteria to be used can be used.

  The amino acid sequences of Aβ4-10, Aβ1-42, and Aβ1-20 are considered to have epitopes, but when administered orally by a microorganism in which the Aβ molecular species according to the present invention is expressed, antibody production mainly Is induced.

  The vector according to the present invention may contain a regulatory element for efficiently expressing the target DNA, for example, a promoter, an enhancer, a transcription terminator, etc., and a plurality of expression units may be linked.

  The vectors according to the invention can be prepared by standard methods well known in the art. For example, JP 2005-500054 and references cited therein describe various appropriate vectors and methods for producing these vectors.

  The inventors of the present application applied a surface expression system for microorganisms exemplified by lactic acid bacteria, in order to carry out oral immunization for the prevention and treatment of Alzheimer's disease more simply and safely. The surface expression system of lactic acid bacteria is to express the target protein on the surface layer of lactic acid bacteria. If this system is used, a protein serving as a vaccine antigen is expressed on the surface of a lactic acid bacterium, and the lactic acid bacterium is orally administered so that cells in charge of intestinal immunity can be recognized. Lactic acid bacteria themselves have been widely eaten as safe food for a long time, and recently they are being actively used as health foods and pharmaceuticals as probiotics. Lactic acid bacteria that are safe and useful for health are used as a host for gene expression. Naru et al. Proved its usefulness as a vaccine by orally administering a vaccine antigen expressed on the surface of lactic acid bacteria (see JP-T-2005-500054). This time, the present inventors have pioneered the development of a safe and economical oral vaccine by applying this method.

  In order to express foreign proteins on the cell surface using bacterial outer membrane proteins, the outer membrane proteins and foreign proteins are linked at the gene level so that the fusion protein can be biosynthesized and stably It must pass through the inner membrane to adhere to and maintain the outer membrane. For this purpose, an outer membrane protein must be selected and used as a motif for surface expression (see S. Y. Lee, J. H. Choi, Z. Xu, Trends Biotechnol., 21, 45 (2003)). Various studies have been conducted on the presentation of proteins on the surface of gram-positive bacteria, SY Lee, JH Choi, Z. Xu, Trends Biotechnol., 21, 45 (2003); P. Samuelson. , E. Gunneriusson, PA Nygren, S. Stahl, J. Biotechnol., 96, 129 (2002); K. Leenhouts, G. Buist, J. Kok, Antonie Van Leeuwenhoek, 76, 367 (1999); E. Gunneriusson , P. Samuelson, M. Uhlen, PA Nygren, S. Stahl, J. Bacteriol., 178, 1341 (1996); S. Stahl, A. Robert, E. Gunneriusson, H. Wernerus, F. Cano, S. Liljeqvist, M. Hansson, TN Nguyen, P. Samuelson, Int. J. Med. Microbiol., 290, 571 (2000); L. Steidler, J. Viaene, W. Fiers, E. Remaut, Appl. Environ. Microbiol , 64, 342 (1998); JC Piard, I. Hautefort, VA Fischetti, SD Ehrlich, M. Fons, A. Gruss, J. Bacteriol., 179, 3068 (1997); K. Savijoki, M. Kahala, A. Palva, Gene, 186, 255 (1997); A. Strauss, F. Gotz, Mol. Microbiol., 21, 491 (1996); and S. Avall-Jaaskelainen, A. Lindholm, A. Palva, Appl. Envir on. Microbiol., 69, 2230 (2003)), it has been pointed out that there are problems with the size of the protein that can be displayed on the surface and the stability of the motif at present.

  On the other hand, Naru et al., As a novel motif, examined the expression of the target protein on the bacterial cell surface using an expression plasmid containing the gene of poly-γ-glutamate biosynthetic enzyme derived from the genus Bacillus, It has been shown that various proteins can be presented on the surface layer of lactic acid bacteria, which are fungi, and Escherichia coli, which is a gram-negative bacterium (Special Table No. 2005-500054).

  In the present invention, the inventors applied the system described in JP 2005-500054 to the expression of Aβ molecular species. In the present invention, in the surface expression system of microorganisms adopted by the inventors, cells involved in the synthesis of poly-γ-glutamic acid derived from Bacillus as a new surface expression carrier for expressing a large amount of foreign protein on the surface of the microorganism By selecting an outer membrane protein and using it, a vector for surface expression that expresses a foreign protein or peptide (in the present invention, Aβ molecular species) on the surface of a microorganism is prepared, and various traits transformed by the vector are used. A foreign protein is efficiently expressed on the surface of the transformant from the transformant.

  To achieve the above object, the present invention provides one or more of pgsA, pgsB and pgsC, which are genes constituting a poly-γ-glutamic acid biosynthetic enzyme (pgs) complex. A surface expression vector is provided. Specifically, the present invention provides a surface expression vector for expressing an Aβ molecular species on the surface of a microorganism. For example, one embodiment of the pgsA gene is homologous to the nucleotide sequence set forth in SEQ ID NO: 1. Contains nucleotide sequence. In addition, a surface expression vector is also provided for expressing an Aβ molecular species protein on the surface of a microorganism, which contains not only the gene encoding the Aβ molecular species but also a transcription stop codon at the 3 'end. Furthermore, cell transformants including lactic acid bacteria exemplified as the optimum embodiment are provided to be transformed using the expression vector.

  The Alzheimer's disease vaccine which expresses the Aβ molecular species according to the present invention on the surface layer, in particular, a microorganism having lactic acid bacteria as an optimal embodiment, as a main aspect, can be used for the treatment and / or prevention of Alzheimer's disease in mammals. Therefore, according to the present invention, a therapeutic / preventive method for treating Alzheimer's disease comprising the administration of a therapeutic / preventive effective amount of the Alzheimer's disease vaccine according to the present invention to a subject, and an agent for treating / preventing Alzheimer's disease. There is provided the use of an Alzheimer's disease vaccine according to the present invention. The administration target is a mammal, for example, a dog, a primate, etc., preferably a human.

  The administration method of the Alzheimer's disease vaccine brought about by the present invention is not particularly limited and a general method can be applied. However, since the lactic acid bacteria serving as the host of the vaccine of the present invention can be administered orally, oral administration is not possible. This is a particularly preferred aspect in that the subject can administer himself. Other examples include intraperitoneal injection, intratracheal injection, intrabronchial injection and direct intrabronchial instillation, subcutaneous injection, transdermal transport, intraarterial injection, intravenous injection, nasal administration, and the like. .

  The amount of Alzheimer's disease vaccine administered can be a therapeutically effective amount, and such amount can be readily determined by one of ordinary skill in the art. The dose is preferably adjusted according to the severity of the disease state, sex, age, weight, etc. of the subject, but such adjustment of the dose can be appropriately performed by a doctor or veterinarian.

  Once the Alzheimer's disease vaccine according to the present invention is administered to a subject, it is expected that the therapeutic action for Alzheimer's disease will continue for a relatively long period of time. In particular, when administered orally, it has been confirmed that antigens are presented in intestinal epithelial cells for a long period of time, and antibody production against this is induced. In view of this point, those skilled in the art can create an appropriate dosing schedule.

  The Alzheimer's disease vaccine according to the present invention can be administered to a subject as a pharmaceutical composition containing lactic acid bacteria as an optimal embodiment. Therefore, according to the present invention, there is provided a pharmaceutical composition for treating / preventing Alzheimer's disease comprising lactic acid bacteria according to the present invention. According to a preferred embodiment of the present invention, the pharmaceutical composition is for oral administration.

  The pharmaceutical composition according to the present invention can be prepared by methods known in the art depending on the administration route and dosage form. For example, dosage forms such as capsules and solutions can be used as pharmaceutical compositions for oral administration. Therefore, the pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier, diluent, preservative and the like according to each dosage form.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to this example.

(Production of Lactic Acid Bacteria Presenting Full-length Aβ Containing Aβ1-42 (abbreviated as Aβ42) on the Cell Surface)
(1) Construction of Aβ42 display vector on lactic acid bacteria cell surface layer First, plasmid pHCEILB-pgsBCA (obtained from BioLeaders Corporation) containing pgsA gene (SEQ ID NO: 1) which is one of poly-γ-glutamic acid biosynthetic enzymes. PCR reaction using a template and oligonucleotides having the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 as primers (MJ Research PTC-200, Peltier Thermal Cycler; the same PCR is used for the following PCR reactions), 94 ° C 5 Min / (94 ℃ ・ 1min / 55 ℃ ・ 1min / 72 ℃ ・ 1min 20sec: 30cycle) / 72 ℃ ・ 10min / 15 ℃ held, NucleoSpin Extract kit (MACHEREY-NAGEL) ) Was used to purify the pgsA gene.

  Next, with reference to the base sequence (SEQ ID NO: 4) encoding Aβ of Homo sapiens Amyloid beta (A4) precursor protein (APP: Accession No. 41406056), a frequently used codon that increases expression efficiency in lactic acid bacteria PCR reaction using oligonucleotides having the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7 as primers for optimization, 94 ° C, 5 minutes / (94 ° C, 1 minute / 50 ° C, 1 minute / 72 ° C., 10 seconds: 15 cycles) / 15 ° C., and a new Aβ42 gene was synthesized (SEQ ID NO: 8). After completion of the reaction, purification was performed using NucleoSpin Extract kit (MACHEREY-NAGEL). Furthermore, PCR reaction using the synthesized Aβ42 gene as a template and oligonucleotides having the nucleotide sequences described in SEQ ID NO: 9 and SEQ ID NO: 10 as primers, 94 ° C. 5 minutes / (94 ° C./1 minute / 55 ° C. 1 Min / 72 ° C./1 minute: 30 cycles) / 72 ° C./10 minutes / 15 ° C. was maintained, and the gene of Aβ42 to which the restriction enzyme sequence was added was amplified (SEQ ID NO: 11). Similar to the previous PCR, after completion of the reaction, the intended Aβ42 gene was purified using NucleoSpin Extract kit (MACHEREY-NAGEL). This amplified gene of Aβ42 is the amplified gene of 145 bp shown in the target SEQ ID NO: 11. Both ends of the synthesized Aβ42 gene are configured so that recognition sites for the restriction enzymes BamHI and XbaI exist with the primers of SEQ ID NO: 9 and SEQ ID NO: 10. The amplified Aβ42 gene is cleaved with restriction enzymes BamHI and XbaI, and is a constituent gene of the vector pHCEIILB-pgsA-L for surface expression of lactic acid bacteria, and a linker sequence 3'- following the pgsA serving as an anchor protein to the cell surface A translational codon is inserted in the terminal site together and ligated by TAKARA Ligation Kit Version 2.1 (Takara Bio), so that the target Aβ42 display vector pHCEIILB-pgsA-L- having pgsA-L-Aβ42 in this order is added. Aβ42 was obtained (FIG. 1).

  This vector was digested with restriction enzymes BamHI and XbaI, Aβ42 gene transfer was confirmed, and its base sequence was confirmed using ABI PRISM (registered trademark) 3100-Avant Genetic Analyzer (Applied Biosystems).

(2) Production of Lactic Acid Bacteria Presenting Aβ42 on the Cell Surface Layer The Aβ42 surface expression vector pHCEIILB-pgsA-L-Aβ42 obtained in (1) above was transferred to an E. coli DH5α competent cell by the heat shock method. Transformed, Luria-Bertain (LB) agar medium (trypton 1 w / v%, yeast extract 0.5 w / v%, sodium chloride 1 w / v%, and agar powder 1.5 w / v containing 500 μg / ml erythromycin %), Transformants were selected. Next, the obtained transformant was cultured in an LB liquid medium, and the cells were collected, and then pHCEIILB-pgsA-L-Aβ42 was purified with Wizeard (registered trademark) Plus SV Miniprepsp (Promega). Next, the obtained surface expression vector pHCEIILB-pgsA-L-Aβ42 was transformed into Lactobacillus casei BLS525 strain by electroporation, and applied on MRS agar medium (Oxoid) containing 20 μg / ml erythromycin. After culturing at 37 ° C. for 48 hours, transformants were obtained. The obtained transformant was cultured in an MRS liquid medium at 37 ° C. for 24 hours, and the cells were collected by centrifugation at 7000 rpm for 10 minutes. The collected cells are washed with PBS buffer solution (pH 7.4), resuspended in the same buffer solution, and then kept on ice. The ultrasonic crusher (output 4, 2 minutes x 2 times / Tomy Seiko Co., Ltd.) ). The crushed liquid was centrifuged at 3,000 rpm for 5 minutes × 3 times to remove impurities, and fractionated by centrifugation at 16,500 rpm for 20 minutes. All cell, cytoplasm and cell membrane fractions were suspended in the sample buffer, heat-denatured at 100 ° C. for 10 minutes, and subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, it was transferred to a PVDF membrane (Millipore). Shake the PVDF membrane with blocking buffer solution (PBS, 5% skim milk, pH 8.0) for 1 hour to block, and then dilute the pgsA polyclonal antibody (obtained from BioLeaders Corporation) 1000 times with blocking buffer as the primary antibody. And allowed to react at 37 ° C. for 12 hours. After the reaction, the membrane was washed with TBST buffer (NaCl 8.8g / 1M Tris-HCl pH8.0 / 0.5ml Tween20 in 1L), and biotinylated anti-rabbit IgG antibody (Vector) was used as the secondary antibody. And diluted 2000 times with blocking buffer and allowed to react for 3 hours. After the reaction, the membrane was washed, avidinized with vectastain ABC kit standard (Vector), further developed with peroxidase substrate kits DAB / Ni substrate (Vector), and expression of Aβ42 fused with pgsA in the cell membrane fraction Was confirmed (FIG. 2). Similarly, Western blotting was performed using a monoclonal antibody against Aβ42 (Chemicon international) and a biotin-labeled anti-mouse IgG antibody (Vector) as a secondary antibody to confirm the expression of Aβ42 on the surface of lactic acid bacteria in the cell membrane fraction. did.

  Subsequently, the surface expression of Aβ was observed by fluorescence-activating cell sorting (Facs) flow cytometry. The expressed cells were washed three times with PBS buffer (pH 7.4) and then resuspended in blocking PBS buffer (pH 7.4). A monoclonal antibody against Aβ was used as a primary antibody and reacted at 4 ° C. for 12 hours. After completion of the reaction, the mixture was washed with PBS buffer, and a biotin-labeled secondary antibody was reacted at 4 ° C. for 3 hours. After completion of the reaction, color was developed with biotin-specific streptavidin-R-picotriene, and the cells were washed twice with PBS buffer and then measured with Facs scan. As a result, it was confirmed that Aβ42 was expressed on the cell surface as compared with the control (FIG. 3).

(Production of Lactic Acid Bacteria Displaying Aβ Molecular Species Containing Aβ1-20 (abbreviated as AβN-20) on the Surface)
(1) Construction of vector for displaying AβN-20 on the surface of lactic acid bacteria cells Site showing the epitope from N-terminal to 20 amino acids of Aβ in the surface expression vector pHCEIILB-pgsA-L similar to the above-mentioned surface expression vector of Aβ42 In order to introduce the oligonucleotide, the oligonucleotides having the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 13 are used as primers using pgsA, linker sequence and Aβ42 gene introduced into pHCEIILB-pgA-L-Aβ42 as templates. 364 bp of the pgsA gene by holding the PCR reaction, 94 ° C 5 min / (94 ° C 1 min / 55 ° C 1 min / 72 ° C 1 min: 30 cycles) / 72 ° C 10 min / 15 ° C From the base sequence of the eyes, a linker sequence and a gene of 20 amino acids from the N-terminus of Aβ42 were amplified. Similar to the PCR in Example 1 described above, after completion of the reaction, the intended AβN-20 gene was purified using NucleoSpin Extract kit (MACHEREY-NAGEL). The gene site amplified at this time was 888 bp in size. The primers of SEQ ID NO: 12 and SEQ ID NO: 13 are configured so that recognition sites for restriction enzymes PstI and XbaI exist.

  From the amplified pgsA gene 363 bp, the linker sequence and the AβN-20 gene are cleaved with restriction enzymes PstI and XbaI, and are the constituent genes of lactic acid bacteria surface expression vector pHCEIILB-pgsA-L, and anchor proteins to the cell surface PgsA is inserted into the 3'-terminal portion of the linker sequence following pgsA together with a translation codon, and ligated by TAKARA Ligation Kit Version 2.1 (Takara) to have pgsA-L-AβN-20 in this order. The target AβN-20 display vector pHCEIILB-pgsA-L-AβN-20 was obtained (FIG. 4). This vector was digested with restriction enzymes PstI and XbaI to confirm gene introduction of AβN-20, and its base sequence was confirmed using ABI PRISM (registered trademark) 3100-Avant Genetic Analyzer (Applied Biosystems).

(2) Preparation of Lactic Acid Bacteria Presenting AβN-20 on the Cell Surface Layer Using the surface expression vector pHCEIILB-pgsA-L-AβN-20 obtained in (1), AβN-20 was prepared in the same manner as in Example 1. Expression was confirmed.

  As shown in FIG. 5, the expression of AβN-20 was confirmed in the cell membrane fraction as a fusion protein with the anchor protein pgsA in the pgsA antibody. Similarly, the expression of AβN-20 as a fusion protein with pgsA was confirmed in the cell membrane fraction of the Aβ monoclonal antibody. Furthermore, surface expression of AβN-20 was also confirmed in Facs scan (FIG. 6).

(Immune experiment by oral administration of Aβ-expressing lactic acid bacteria)
The immunogenicity by oral administration of lactic acid bacteria expressing Aβ constructed in Examples 1 and 2 on the surface was examined.

(1) Lactic acid bacteria administration method Lactic acid bacteria transformed with pHCEIILB-pgsA-L-Aβ42 (Aβ42 expression vector) obtained in Example 1 or obtained in Example 2 using 6 mouse C57BL6 strains per group as inoculated animals The lactic acid bacteria transformed with pHCEIILB-pgsA-L-AβN-20 (AβN-20 expression vector) were orally administered. The dosage is 1 × 10 ⁹ cfu / ml (3-1), 1 × 10 ¹⁰ cfu / ml (3-2) or 5 × 10 ¹⁰ cfu / ml (3-3) for Aβ42 expression vector, AβN The -20 expression vector was 1 × 10 ⁹ cfu / ml (2-1) or 1 × 10 ¹⁰ cfu / ml (2-2). As a control, the same lactic acid bacteria transformed with pHCEIILB-pgsA-L (control vector that does not express Aβ) were orally administered to the mice at a dose of 1 × 10 ¹⁰ cfu / ml (1-1).

Specifically, the following procedure was performed. The various transformants of the lactic acid bacteria were precultured at 37 ° C. and then cultured to 1 × 10 ^{9 to} 5 × 10 ¹⁰ cfu / ml. Bacteria were collected by centrifugation, washed with PBS, and killed. Subsequently, the dead cells were pulverized through a freeze-drying process. The powdered cells were resuspended in PBS to a concentration of 1 × 10 ^{9 to} 5 × 10 ¹⁰ cfu / ml. 100 μl of this suspension was orally administered to mice at the doses indicated above. The administration period was continuous administration for 6 days within 1 week and continued for 4 weeks. In order to confirm the production of antibodies against Aβ in the blood, blood samples were collected on a total of 5 blood sampling schedules before the start of the experiment, 3 weeks, 5 weeks, 6 weeks, and 7 weeks after the start of administration. Got. The obtained serum sample was stored at −20 ° C. until measurement.

(2) Measurement of antibody titer in blood Antibody titer in serum was measured by the following ELISA method. 42 amino acid residues of Aβ and 16 amino acid residues from the N-terminal side were synthesized. Each synthesized peptide was added to a 96-well plate at a concentration of 500 ng / 100 μl / well (bicarbonate buffer: pH 9.6) and immobilized at 4 ° C. for 12-14 hours. The plate immobilized with each synthetic peptide was washed 5 times with 300 μl of PBST. Blocked with 10% skim milk for 2 hours at room temperature. The plate was washed again 5 times with 300 μl PBST. Positive standard, negative control and mouse serum diluted with 2% skim milk (1:50) were added at 100 μl / well, respectively, and reacted at 37 ° C. for 1 hour. The plate was washed again 5 times with 300 μl PBST. Peroxidase-conjugated anti-mouse IgG diluted with 2% skim milk (1: 1000) was added to the plate at 100 μl / well and allowed to react at 37 ° C. for 1 hour. The plate was washed again 5 times with 300 μl PBST. Substrate OPD was added at 100 μl / well and allowed to react for 15-20 minutes in the dark at room temperature. 2M H ₂ SO ₄ was added at 50 μl / well to stop the reaction, and the absorption at a wavelength of 450 nm was measured.

  The results are shown in FIGS. As is apparent from FIGS. 7 and 8, lactic acid bacteria in which the peptides of AβN-20 and Aβ42 were expressed on the surface were orally administered to mice, and the antibody elevation in the serum was measured by ELISA. As a result, AβN-20 In the lactic acid bacteria-administered group in which each peptide of Aβ42 was expressed, a significant increase in serum antibody was observed as compared to the control group. From this result, it became clear that antibodies against Aβ can be induced by orally administering lactic acid bacteria expressing Aβ molecular species to the surface of the cells produced by the present inventors.

  The present invention provides a vaccine for the prevention / treatment of Alzheimer's disease that can be provided for the prevention / treatment of Alzheimer's disease and that can be used in a safe and simple administration form with few side effects. If the Alzheimer's disease vaccine using lactic acid bacteria which is the optimal embodiment according to the present invention is used, it becomes possible to take preventive and therapeutic drugs for Alzheimer with the same feeling and convenience as conventional foods of lactic acid bacteria. Considering that this disease is particularly common among the elderly, oral administration that can be ingested easily and the realization of low cost will bring great benefits to society from the perspective of improving patient quality of life and the medical economy in the aging society. It can be said that the invention is given.

[0005]
Means that a child has been inserted.
[0021]
It is considered that there are a plurality of humoral immunity induction epitopes of Aβ. To prepare a fragment containing the epitope, for example, if the epitope is present in the region of 4th to 10th amino acids of Aβ (Aβ4-10) The expression vector for producing them on the surface of the microorganism will contain a gene encoding Aβ4-10.
[0022]
The nucleotide sequence of DNA encoding this amino acid sequence is not particularly limited, and examples thereof include the 12th to 30th nucleotides in the nucleotide sequence represented by SEQ ID NO: 4. In addition, by using codons having the same amino acid sequence but different base sequences, the expression level can be increased by using a codon preferred by a microorganism such as lactic acid bacteria to be used.
[0023]
According to a preferred embodiment of the present invention, the antigen peptide fragment comprises the first to 42 amino acids (Aβ1-42) of the Aβ peptide. The amino acid sequence of Aβ1-42 is exemplified by the amino acid sequence represented by SEQ ID NO: 5, and therefore the antigen peptide fragment includes the amino acid sequence represented by SEQ ID NO: 5. The nucleotide sequence of DNA encoding this amino acid sequence is not particularly limited, and examples thereof include the nucleotide sequence represented by SEQ ID NO: 4. Further, by using codons having the same amino acid sequence but different base sequences, codons preferred by microorganisms such as lactic acid bacteria to be used can be used.
[0024]
According to another preferred embodiment of the present invention, the antigen peptide fragment comprises amino acids 1 to 20 (Aβ1-20) of Aβ peptide. Examples of the amino acid sequence of Aβ1-20 include those containing amino acids 1 to 20 in the amino acid sequence represented by SEQ ID NO: 5. The nucleotide sequence of DNA encoding this amino acid sequence is not particularly limited, and examples thereof include those containing the first to 60 nucleotides in the nucleotide sequence represented by SEQ ID NO: 4. Further, by using codons having the same amino acid sequence but different base sequences, codons preferred by microorganisms such as lactic acid bacteria to be used can be used.
[0025]
The amino acid sequences of Aβ4-10, Aβ1-42, and Aβ1-20 are considered to have epitopes, but when administered orally by a microorganism in which the Aβ molecular species according to the present invention is expressed, antibody production mainly Is induced.

Claims (14)

  An expression vector for producing amyloid β molecular species on the surface of a microorganism.   2. A gene encoding one or more genes selected from pgsA, pgsB and pgsC encoding a poly-γ-glutamic acid biosynthetic enzyme complex and a gene encoding amyloid β molecular species. An expression vector for producing the described amyloid β molecular species.   The amyloid β molecular species according to claim 2, wherein one or more genes selected from pgsA, pgsB and pgsC are derived from a Bacillus strain that produces a poly-γ-glutamic acid biosynthetic enzyme complex. An expression vector for production.   The amyloid β molecular species is selected from a polypeptide comprising an amino acid sequence up to the 42nd amino acid sequence from the N-terminal sequence of amyloid β and a polypeptide comprising an amino acid sequence up to the 20th amino acid sequence from the N-terminal sequence of amyloid β. An expression vector for producing the amyloid β molecular species according to claim 3.   The expression vector for producing the amyloid β molecular species according to any one of claims 1 to 4, wherein the microorganism is a lactic acid bacterium.   A lactic acid bacterium transformed with an expression vector for producing amyloid β molecular species on the surface of a microorganism.   The transformed lactic acid bacterium according to claim 6, wherein the expression vector for producing amyloid β molecular species on the surface of the microorganism is the expression vector according to claims 1 to 5.   The transformed lactic acid bacterium according to claim 6 or 7, wherein the lactic acid bacterium belongs to the genus Lactobacillus.   A method for inducing an anti-amyloid β antibody by administering a microorganism expressing an amyloid β molecular species to an individual.   The method for inducing an anti-amyloid β antibody according to claim 9, wherein a microorganism expressing the amyloid β molecular species is orally administered to an individual.   The method according to claim 9 or 10, wherein the microorganism expressing amyloid β molecular species is the lactic acid bacterium according to any one of claims 6 to 8.   A method for preventing and / or treating Alzheimer's disease, which comprises inducing immunity to an amyloid β molecular species in an individual by administering a microorganism expressing the amyloid β molecular species to the individual.   The method for preventing and / or treating Alzheimer's disease according to claim 12, wherein the microorganism expressing the amyloid β molecular species is the lactic acid bacterium according to any one of claims 6 to 8.   A vaccine for the prevention / treatment of Alzheimer's disease comprising the lactic acid bacterium according to any one of claims 6 to 8 as a main component.

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